Recombinant Mouse Mast Cell Protease-11/Prss34 Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Mast Cell Protease-11/Prss34 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a colorimetric peptide substrate, N-carbobenzyloxy-Arg-ThioBenzyl ester (Z-R-SBzl), in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The specific activity is >20,000 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Mast Cell Protease-11/Prss34 protein
  Met20-Ser318, with a C-terminal 10-His tag
• Accession #: Q80UR4
• N-terminal Sequence: Met20
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Prss34
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 34 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 48 kDa doublet, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, pH 7.5 (TCN)
  • Assay Buffer: 50 mM Tris, pH 8.0
  • Recombinant Mouse Mast Cell Protease‑11/Prss34 (rmMCP-11) (Catalog # 2857-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
  • Substrate: Z-Arg-SBzl (SM Biochemicals, Catalog # SMSB01), 10 mM in DMSO
  • 5,5'Dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rmMCP-11 to 200 µg/mL with Activation Buffer.
  2. Dilute Thermolysin to 0.4 µg/mL with Activation Buffer.
  3. Mix equal volumes of 200 µg/mL rmMCP-11 and 0.4 µg/mL Thermolysin for final concentrations of 50 µg/mL and 0.2 µg/mL, respectively.
  4. Incubate at 37 °C for 30 minutes.
  5. Stop the reaction with 10 mM 1,10 Phenanthroline.
  6. Dilute activated rmMCP-11 to 0.1 ng/µL in Assay Buffer.
  7. Dilute substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
  8. Load 50 µL of the 0.1 ng/µL rmMCP-11 into plate, and start the reaction by adding 50 µL of the substrate/DTNB mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL substrate mix without any rmMCP-11.
  9. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
  10. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 13260 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rmMCP-11: 0.005 µg
  • DTNB: 100 µM
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Mast Cell Protease-11/Prss34 Protein, CF

• EOS
• Mast Cell Protease11
• Mast Cell Protease-11
• MCP-11
• MCPT11
• Prss34

Background

Mast Cell Protease-11 (MCP-11) is encoded by Prss34, one of 13 genes on mouse chromosome 17A3.3 that correspond to functional trypsin-like serine proteases (1). The deduced amino acid sequence of mouse MCP-11 consists of 318 residues with a signal peptide (residues 1 to 19), a pro region (residue 20 to 34), and a catalytic domain (35 to 318). The mRNA is preferentially expressed in spleen and bone marrow. The mouse MCP-11 (residues 20 to 318) was expressed in the NS0 cells with a foreign signal peptide. After being treated with thermolysin, the purified enzyme is active against a peptide substrate described in the Activity Assay Protocol. Apparently, the human gene corresponding to Prss34 encodes a protein that is not enzymatically active due to a mutation that leads to a premature translation termination codon.

1. Wong, G.W. et al. (2004) J. Biol. Chem. 279:2438.

Bioinformatics

• Gene Symbol: Prss34
• Uniprot:
  • Q80UR4()